Artificial insemination (AI) is a technique wherein spermatozoa are placed into an animal's uterus or cervix by artificial means rather than by natural copulation. It is widely used as a method of mating, and in breeding of animals to propagate desirable characteristics, particularly in the case of farm animals such as cattle, swine, sheep, poultry and horses, but also in case of pets such as pedigree dogs, aquatic animals or endangered species.
Usually spermatozoa is collected, extended and then preserved by e.g. cryopreservation. The use of cryopreservation techniques presupposes that the spermatozoa from the specific species of animal tolerate such treatment without resulting in too much deterioration of the spermatozoa quality, viability and fertilization capacity. The spermatozoa are then transported to the female's location either cryopreserved or freshly stored, which ever is suitable. It is vital that the spermatozoa are maintained viable until the time of insemination and for a sufficient period of time inside the female animal after insemination until the egg cell(s) reach the location of fertilization.
Artificial insemination of farm animals has been used since the 1940s and is now widely used in the agriculture industry, especially for breeding dairy cattle and swine. An overview over the development of modern AI and the challenges of the breeders as regards the use of artificial insemination and preservation of spermatozoa is disclosed in R. H. Foote (2002), American Society of Animal Science (http://www.asas.org/symposia/esupp2/Footehist.pdf) and in “Reproduction in farm animals”, edited by B. Hafez, E. S. E. Hafez.—7th ed., Philadelphia, Lippincott Williams & Wilkins, 2000.—XIII, ISBN 0-683-30577-8 (ib.). Artificial insemination has become an important economical means for breeding animals in agricultural industry, both as regards breeding of animals with specific preferred genetic characteristics and animal production in general.
However, there are some limitations in respect of obtaining pregnancies in the female animal by performing artificial insemination. For example, the shelf life and viability of the collected spermatozoa, both during storage, after thawing in case of cryopreserved spermatozoa, and after insemination, are essential for a successful breeding result. Whether suitable preservation techniques are available for the specific species of animals vary. For cattle, cryopreservation techniques are widely used. On the other hand, spermatozoa from other species such as swine are less tolerant to cryopreservation techniques, resulting in less flexibility as regards semen processing and storing possibilities.
Furthermore, to obtain spermatozoa with suitable fertilizing capacity, it is desired that the preservation method used also provides for maintenance of the fertilizing capacity after insemination. There has been a lot of focus and research for preservation methods aiming at providing storage methods and means which ensure that the spermatozoa maintain the fertilizing capacity for a longer time after collection and till the point of insemination.
If the shelf life is short as regards maintaining fertilizing capacity after insemination, it is more difficult to meet the preferred point of insemination with respect of ovulation. In case of short shelf life characteristic, good preservation techniques for spermatozoa that provide for longer shelf life and thus longer fertilizing capacity are vital. There is yet no preservation method available that provides sufficient shelf life and spermatozoa viability in a sufficient period after insemination to meet the breeders need for flexibility, especially when there is a long and time consuming transport distance between the location of the male and thus the place where semen collection is performed, and the female recipient.
Furthermore, a preservation method providing a more controlled and long-lasting availability of the spermatozoa would reduce the need for artificially provoked ovulation by hormone treatment. This would be beneficial both economically, according to consumer demands, and in respect of animal health.
Thus, there is a need for methods ensuring that the spermatozoa maintain the fertilizing capacity for a longer time after insemination, e.g. maintain the fertilizing capacity for a longer period when placed inside the female recipient.
At present, artificial insemination (AI) in cattle is widely performed using cryopreserved spermatozoa. Cryopreserved spermatozoa can be stored in liquid nitrogen for decades until used. However, when spermatozoa are thawed, AI has to be performed within a few hours. After insemination the cryopreserved spermatozoa have fertilizing potential for approximately 12-24 hours, and AI has to be performed within approximately 12-24 hours before ovulation. Thus, there is a need for preservation techniques for breeding of cattle providing spermatozoa which have sufficient shelf life characteristics and which preferably maintain fertilizing capacity for several days.
In some countries, liquid stored bull spermatozoa are used in order to reduce the number of sperm cells per AI dose. The spermatozoa have fertilizing capacity for approximately 24-36 hours before insemination. AI has to be performed within approximately 24 hours after spermatozoa collection. However, liquid preserved bull spermatozoa are encumbered with several drawbacks such as shortened/reduced shelf life and reduced scope of distribution.
In swine, cryopreserved spermatozoa are used only for special purposes like export, long distance shipment and for control of contagious diseases. AI in swine is usually performed with liquid preserved semen. The storage time for liquid preserved semen (spermatozoa) will depend on which extender is used. Spermatozoa diluted with short term extenders preserve the fertilizing capacity for approximately 2-3 days, while spermatozoa diluted with long term extenders may preserve the fertilizing capacity for up to 5-6 days. After insemination, fertilizing capacity of the spermatozoa lasts for approximately 12-24 hours. Most sows are inseminated twice during heat with approximately 24 hours interval. With a more flexible system providing longer storage time before insemination (e.g. one week) and/or prolonged storage and release of spermatozoa after insemination (e.g. more than 24 hours), the breeding industry would have more efficient production and distribution, and the breeders would have less need for accuracy in timing of insemination relative to ovulation.
In horse-breeding the breeder is mostly dependent on fresh spermatozoa due to lack of applicable preservation techniques for horse spermatozoa. This is a large problem in the horse-breeding and horse racing industry since the preferred stud for a specific brood mare most often is situated in a different country, requiring long transport time. In addition, due to the lack of suitable preservation methods for horse spermatozoa, AI of fresh horse spermatozoa must be performed within 24 hours after the spermatozoa collection. Horse breeding is thus attended with an undesired time pressure which often results in reduced sperm quality or reduced impregnation due to incorrect insemination in respect of ovulation.
Thus, there is a need for preservation methods that ensure both longer shelf life in respect of necessary delivery transport time and a longer shelf life after insemination. An applicable preservation method for horse semen would be of great economic and practical value, and would possibly revolutionize the horse breeding industry.
A spermatozoa preservation system that provides sufficient viability both during storage, before and after insemination would be beneficial for the breeding industry in general. A more flexible preservation system would render the breeding work for all species of animals easier and result in an increase in successful impregnation. A system where the breeder is less dependent on meeting the most preferable insemination point in time in respect of ovulation provides more flexibility.
In order to extend the fertilizing capacity of ejaculated spermatozoa, several preservation methods including cryopreservation and liquid preservation have been investigated.
Several studies of storage of spermatozoa within capsules have been published. These studies have used encapsulation methods that result in a particle where the spermatozoa are located within a liquid core surrounded by a semipermeable membrane.
Nebel et al. (1985), Microencapsulation of bovine spermatozoa. J. Anim. Sci. 1985 60(6):1631-39, describes a method for encapsulation of bovine spermatozoa in capsules made of alginate in combination with polylysin. This method is based on a previously published method from Lim and Sun (1980), Microencapsulated islets as bioartificial endocrine pancreas. Science 1980 210(4472):908-10, which was working with encapsulated insulin production from encapsulation of langerhans cells. Nebel et al. (1985), reported results both from storage at 37° C. and 335 inseminations, and results for encapsulated spermatozoa was compared to un-encapsulated control samples. No major differences between encapsulated and control samples was reported in this study. Other published studies has also demonstrated that spermatozoa can be encapsulated within capsules with similar methods, and maintain their functionality in vivo (Munkittrick et al. (1992) Accessory sperm numbers for cattle inseminated with protamine sulfate microcapsules. J. Dairy Sci., 75(3):725-31 (Bovine), Vishwanath et al. (1997) Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers. Theriogenology, 48: 369-76 (Bovine) and Maxwell et al. (1996). Survival and fertility of micro-encapsulated ram spermatozoa stored at 5° C. Reprod. Dom. Anim., 31:665-73(Sheep). Munkittrick et al. (1992) and Vishwanath et al. (1997) report however, that encapsulated spermatozoa are not as efficient as untreated spermatozoa when inseminated at the same time. This was explained with that encapsulated spermatozoa might need some time to be released from the capsules before fertilization can occur.
Conte et al. (1998), in EP 0922 451 B1 issued to Universitá di Pavia and Universitá Degli Studi Di Milano and Torre et al. (2002), Boar semen controlled delivery system: storage and in vitro spermatozoa release. J. Control. Release, 85:83-89, have developed an alternative method for encapsulation of boar spermatozoa. In this method, the spermatozoa are added to a solution containing calcium or barium, and this suspension is dripped into a solution containing alginate. A capsule of calcium or barium alginate is formed spontaneously round the drop as it hits the alginate solution. This method is claimed to be gentler to the cells compared to the method described by Nebel et al. (1985). Another advantage is that this method implies very little dilution of the spermatozoa solution, which is claimed to be beneficial for the viability of the cells.
Faustini et al. (2004), Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18° C., Theriogenology, 61(1):173-184 reports a significant larger fraction of spermatozoa with intact acrosome, and less leakage of enzymes from spermatozoa stored encapsulated with this method compared to untreated spermatozoa stored under the same conditions.
In a recent paper Weber et al (2006), Design of high-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa. Journ. Biotechnol., 123:155-163, also describes a novel system for encapsulation of bovine spermatozoa. This system is designed for high-throughput manufacture of encapsulated spermatozoa using Ca-alginate or cellulose sulfate poly-diallyldimethyl ammonium chloride (pDADMAC) capsules.
Finally, Chou et al., U.S. Pat. No. 6,596,310 B1 (2003) discloses a method of artificial insemination by timed release of sperm from capsules or solid beads, wherein the sperm is maintained in a non-capacitated stage due to the use of an energy source that does not support capacitation.